Coupling Pharmacogenetics and RNA-Seq to Identify Hsp70-dependent Gene Networks
Approximately 26 million Americans are afflicted with Type 1 or Type 2 diabetes and about 60-70% of these individuals develop diabetic peripheral neuropathy (DPN). To date, many approaches toward treating DPN have focused on inhibiting pathogenic targets/pathways that are considered central effectors for mediating the pathophysiological progression of DPN. However, there has been limited translational success of this approach due, at least in part, to differences in the temporal and/or biochemical uniformity by which these targets/pathways contribute to the progression of DPN between individuals. In contrast, we have developed an innovative therapeutic strategy to improve myelinated/unmyelinated fiber function in DPN that does not rely on inhibiting a specific pathogenic mechanism of disease development but is based on pharmacologic induction of cytoprotective molecular chaperones. Heat shock proteins 90 and 70 (Hsp90, Hsp70) are molecular chaperones that are essential for folding proteins into their biologically active structures and for refolding of aggregated/damaged proteins. Numerous conditions that promote cell stress lead to an Hsp90-dependent induction of the heat shock response, a transcriptional upregulation of antioxidant genes and Hsp70. Importantly, pharmacologic inhibition of Hsp90 mimics heat shock and we have synthesized KU-32 as a proprietary, non-toxic, orally bioavailable, small molecule inhibitor of Hsp90. KU-32 treatment reverses multiple clinical indices of insensate DPN and this temporally correlates with an improvement in mitochondrial bioenergetics of adult sensory neurons. Although KU-32 binds and inhibits Hsp90, downstream induction of Hsp70 is required for drug efficacy since KU-32 cannot reverse insensate DPN nor improve mitochondrial bioenergetics in diabetic Hsp70 knockout (KO) mice. Given the broad role of Hsp70 in affecting protein folding, stability and clearance, our hypothesis is that multiple genes and gene networks within sensory neurons and SCs contribute to the reversal of insensate DPN in an Hsp70-dependent manner. High throughput transcript sequencing (RNA-Seq) provides a digital readout of gene expression since it is based on counting reads (# of sequence events), a direct measure of transcript abundance. RNA-Seq has a greater sensitivity and a larger dynamic range then gene microarrays and allows the identification and quantification of transcripts that may not be readily detected by microarrays. Since RNA-Seq is emerging as the favored approach for quantitative profiling of transcriptomes, our aim is to couple the read depth provided by RNA-Seq analysis with the lack of a pharmacologic phenotype in the KU-32 treated diabetic Hsp70 KO mice. Through this approach, we have a robust pharmacogenetic model to identify the breadth of Hsp70-dependent genes/gene networks in sensory neurons and SCs that contribute to the reversal of DPN. This data will provide new leads that underlie drug efficacy and guide the development of mechanistic studies in animal models to aid the translational validation of modulating molecular chaperones as a viable therapeutic approach in humans.

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